Preservative composition for biological samples

Abstract

The purpose of the present invention is to provide a preservative composition which is used in place of or in combination with a cell-penetrating substance such as DMSO that has been commonly used in a culture medium for cryopreservation use and a liquid for cryopreservation use to reduce the toxicity of the cell-penetrating substance and improve the cryopreservation effect of the cell-penetrating substance.　Provided are: a zwitterionic polymer represented by general formula (1), or a labelled product of the zwitterionic polymer; and a preservative composition for biological samples, which contains the zwitterionic polymer or the labelled product. (In the formula, X1 and X2 may be the same as or different from each other, and each independently represent a carbon atom or a nitrogen atom; Y1 and Y2 may be the same as or different from each other, and each independently represent an anion selected from -COO-, -SO3 -, -OP=O(H)O-, -OP=O(CH3)O- and -OP=O(OR1)O-; Z represents a hydrogen atom, an aromatic hydrocarbon group having 6 to 10 carbon atoms in which an alkyl group may be substituted, a 5- to 6-membered aromatic heterocyclic group in which an alkyl group may be substituted, a nitrogenated heterocyclic ammonium salt in which an alkyl group may be substituted, a tetraalkyl ammonium salt, a tetraphenyl phosphonium salt, a tetraalkyl phosphonium salt, a trialkyl sulfonium salt, or a linear or branched alkyl group having 1 to 22 carbon atoms which may have 1 to 3 oxygen atoms in a molecular chain thereof; e and f each independently represent an integer of 0 or 1; l, m and n are numerical values respectively representing the content ratios of repeating units corresponding thereto and respectively satisfying the formulae 0 < l ≤ 1, 0 ≤ m < 1, 0 ≤ n < 1 and l+m+n = 1; and p, q, r, s and t each independently represent an integer of 0 to 6.)

Description

Preservative composition for biological samples

[0001] The present invention relates to a preservative composition for biological samples such as cells.

[0002] Dimethyl sulfoxide (DMSO) and glycerol are widely used as cryopreservatives for cell cryopreservation and are the most effective reagents for protecting cells and organelles. These cryopreservatives protect cells by suppressing the growth of ice crystals (ice crystals) formed within cells during cell freezing. DMSO and other cryopreservatives are cell membrane permeable, slowing the rate of ice crystal growth inside and outside cells and inhibiting ice crystal formation. However, DMSO and glycerol are toxic and are known to cause hypertension, nausea, and vomiting when infused into recipients along with cells or when handled by cell handlers. Furthermore, when DMSO is used as a cryopreservative, fetal bovine serum (FBS) or bovine serum albumin (BSA) is often added. However, because these are derived from animals, there is a problem of quality variation between batches. The present inventors have proposed aprotic zwitterions as substances that can be used in place of DMSO and other commonly used additives to culture media, and have proposed cryopreservation media (Patent Document 1). Furthermore, when a poorly water-soluble substance is added to a culture medium in a cell-based chemical assay, the poorly soluble substance is dissolved in a solvent such as DMSO and then dispersed in the culture medium. DMSO is considered a highly soluble solvent, but it is known that some poorly soluble substances are insoluble even in DMSO. The present inventors have previously proposed the use of aprotic zwitterions as a substance for dissolving poorly soluble substances (Patent Document 2).

[0003] On the other hand, zwitterionic polymers have been used for the purpose of surface modification of biocompatible polymers (Patent Document 3), and have also attracted attention as industrial materials. Various studies on the structure and properties of zwitterions have been conducted from an academic perspective (Non-Patent Documents 1 to 3).

[0004] International Publication No. 2020 / 230721 JP 2018-191623 A JP 2015-213755 A

[0005] Eur. Polym. J. Vol. 26, No. 4, p. 415-421 (1990). ACS Appl. Mater. Interfaces 2018, 10, 17771-17783Chem Asian J. 2021, 16, 1897-1900

[0006] The present inventors have produced aprotic zwitterions as substances that can be used in place of DMSO and other substances that have been widely used as additives to culture media, and have proposed their use in cryopreservation media and as solubilizers for poorly soluble substances, but these have not yet demonstrated sufficient effectiveness. An object of the present invention is to provide a zwitterionic polymer that can be used as an additive to biological samples and as a solubilizer for poorly soluble substances, such as DMSO. A further object of the present invention is to provide a preservative composition for biological samples that, when used in combination with a cell-permeable substance, reduces the toxicity of the cell-permeable substance and further enhances the cryopreservation effect.

[0007] Therefore, the present inventors have investigated the effects of various compounds on the cryopreservation of biological samples such as cells, and have found that by using a zwitterionic polymer having a specific structure, the preservation properties of biological samples such as cells can be improved directly or indirectly by increasing the osmotic pressure through the addition of a water-soluble compound such as an electrolyte and adjusting the solubility and dispersibility of the zwitterionic polymer, and further that it is possible to dissolve poorly soluble substances, thereby completing the present invention.

[0008] The present invention provides the following inventions [1] to

[15] : [1] A zwitterionic polymer represented by the following general formula (1) or a labeled product thereof:

[0009]

[0010] (In the formula, X 1 and X 2 may be the same or different and represent a carbon atom or a nitrogen atom; Y 1 and Y 2 may be the same or different, -COO - , -SO3 - , -OP=O(H)O -, -OP=O(CH3)O - and -OP=O(OR 1 ) O - Z represents a hydrogen atom, an aromatic hydrocarbon group having 6 to 10 carbon atoms which may be substituted with an alkyl group, a 5- to 6-membered aromatic heterocyclic group which may be substituted with an alkyl group, a nitrogen-containing heterocyclic ammonium salt which may be substituted with an alkyl group, a tetraalkylammonium salt, a tetraphenylphosphonium salt, a tetraalkylphosphonium salt, a trialkylsulfonium salt, or a linear or branched alkyl group having 1 to 22 carbon atoms which may have 1 to 3 oxygen atoms in the molecular chain; e and f each represent an integer of 0 or 1; l, m, and n each represent a number indicating the content ratio of each repeating unit, and are numbers which satisfy the conditions 0<l≦1, 0≦m<1, 0≦n<1, and l+m+n=1; p, q, r, s, and t each represent an integer of 0 to 6. [2] Y 1 and Y 2 may be the same or different, -COO - and -SO3 - [3] The zwitterionic polymer or labeled product thereof according to [1], wherein Z is an anion selected from the group consisting of an imidazolyl group, a pyridyl group, pyridinium chloride, a C1-C22 alkylpyridinium chloride, imidazolinium chloride, a C1-C22 alkylimidazolinium chloride, pyridinium bromide, a C1-C22 alkylpyridinium bromide, imidazolinium bromide, and a C1-C22 alkylimidazolinium bromide. [4] A preservative composition for a biological sample, comprising a zwitterionic polymer represented by the following general formula (1) or a labeled product thereof:

[0011]

[0012] (In the formula, X 1 and X 2 may be the same or different and represent a carbon atom or a nitrogen atom; Y 1 and Y 2 may be the same or different, -COO - , -SO3 - , -OP=O(H)O- , -OP=O(CH3)O - and -OP=O(OR 1 ) O - Z represents a hydrogen atom, an aromatic hydrocarbon group having 6 to 10 carbon atoms which may be substituted with an alkyl group, a 5- to 6-membered aromatic heterocyclic group which may be substituted with an alkyl group, a nitrogen-containing heterocyclic ammonium salt which may be substituted with an alkyl group, a tetraalkylammonium salt, a tetraphenylphosphonium salt, a tetraalkylphosphonium salt, a trialkylsulfonium salt, or a linear or branched alkyl group having 1 to 22 carbon atoms which may have 1 to 3 oxygen atoms in the molecular chain; e and f each represent an integer of 0 or 1; l, m, and n each represent a number indicating the content ratio of each repeating unit, and are numbers which satisfy the conditions 0<l≦1, 0≦m<1, 0≦n<1, and l+m+n=1; p, q, r, s, and t each represent an integer of 0 to 6. [5] Y 1 and Y 2 may be the same or different, -COO - and -SO3 -[6] The preservative composition for a biological sample according to [4], wherein Z is an anion selected from the group consisting of an imidazolyl group, a pyridyl group, pyridinium chloride, a C1-C22 alkylpyridinium chloride, imidazolinium chloride, a C1-C22 alkylimidazolinium chloride, pyridinium bromide, a C1-C22 alkylpyridinium bromide, imidazolinium bromide, and a C1-C22 alkylimidazolinium bromide. [7] The preservative composition for a biological sample according to any one of [4] to [6], further comprising one or more water-soluble compounds selected from electrolytes, betaine, zwitterions, alcohols, polyhydric alcohols, and sugars. [8] The preservative composition for a biological sample according to any one of [4] to [7], further comprising a cell-permeable substance. [9] The preservative composition for a biological sample according to any one of [4] to [8], which is a cryopreservative composition for a biological sample, a composition for culturing a biological sample, a medium composition for a biological sample, a composition for preserving a biological sample, a composition for maintaining the function of a biological sample, or a composition for functional testing of a biological sample.

[10] A solubilizer for a poorly soluble substance, comprising the zwitterionic polymer or a labeled form thereof according to any one of [1] to [3].

[11] A method for preserving a biological sample, comprising contacting a biological sample with a composition containing the zwitterionic polymer or a labeled form thereof according to any one of [1] to [3].

[12] The method for preserving a biological sample according to

[11] , wherein the composition containing the zwitterionic polymer or a labeled form thereof according to any one of [1] to [3] further contains one or more water-soluble compounds selected from electrolytes, betaine, zwitterions, alcohols, polyhydric alcohols, and sugars.

[13] The method for preserving a biological sample according to

[11] or

[12] , wherein the composition containing the zwitterionic polymer or a labeled form thereof according to any one of [1] to [3] further contains a cell-permeable substance.

[14] The method for preserving a biological sample according to any one of

[11] to

[13] , wherein the preservation of the biological sample is cryopreservation of the biological sample, culturing of the biological sample, culture medium for the biological sample, maintenance of the function of the biological sample, or functional testing of the biological sample.

[15] A method for dissolving a poorly soluble substance, comprising dissolving the poorly soluble substance in a composition containing the zwitterionic polymer or a labeled form thereof according to any one of [1] to [3].

[0013] The use of the preservative composition for biological samples of the present invention can protect biological samples such as cells, increasing their survival rate even after thawing after cryopreservation. Furthermore, the use of the preservative composition of the present invention can improve the preservation and maintenance of biological materials. Furthermore, the use of the zwitterionic polymer of the present invention can dissolve various poorly soluble substances.

[0014] VimC3C (DMSO-) 1 The H-NMR chart of poly(VimC3C)(methanol) is shown below. 1 The H-NMR chart is shown below. 1 The H-NMR chart of Poly(VpyCC)(methanol-d6) is shown below. 1 The H-NMR chart of Poly(VpyCS)(DO) is shown below. 1 The H-NMR chart of VimCS (DMSO) is shown below. 1 The H-NMR chart of Poly(VimCS)(DO) is shown below. 1 The H-NMR chart of Poly(VpyC3C)50(CH3OH-d) is shown below. 1 The H-NMR chart of Poly(VpyC3C)40(CH3OH-d) is shown below. 1 The H-NMR chart of Poly(VpyC3C)30(CH3OH-d) is shown below. 1 The H-NMR chart of Poly(VpyC3C)20(CH3OH-d) is shown below. 1 The H-NMR chart of Poly(VpyC3C)10(CH3OH-d) is shown below. 1 The H-NMR chart of Poly(VpyCS)40 (methanol-NaCl) is shown below. 1 The H-NMR chart of Poly(VimC3C-co-C8Vim) is shown below. 1H-NMR charts are shown. The relationship between zwitterionic polymer concentration and cell viability after cryopreservation is shown. The relationship between the concentration of NaCl aqueous solution added to the zwitterionic polymer and cell viability after cryopreservation is shown. The relationship between cell viability after cryopreservation depending on the type of solute added to the zwitterionic polymer is shown. The toxicity of zwitterionic polymers to cells is shown. DLS charts are shown for NaCl solutions of zwitterionic polymers. GPC results are shown for zwitterionic polymers. The relationship between the catalyst / monomer ratio during zwitterionic polymer production and cell viability after cryopreservation is shown. The left side of the figure shows data for aqueous zwitterionic polymer solutions, and the right side shows data for zwitterionic polymer medium solutions. The relationship between the effect of NaCl concentration on the viability of K562 cells after cryopreservation with zwitterionic polymer (polyVimC3C) is shown. The relationship between the effect of NaCl concentration on the viability of C6 cells after cryopreservation with zwitterionic polymer (polyVimC3C) is shown. The relationship between NaCl concentration and viability of OVMANA cells after cryopreservation with a zwitterionic polymer (polyVimC3C) is shown. The relationship between NaCl concentration and proliferation rate of C6 cells after cryopreservation with a zwitterionic polymer (polyVimC3C) is shown. The relationship between NaCl concentration and proliferation rate of OVMANA cells after cryopreservation with a zwitterionic polymer (polyVimC3C) is shown. Microscopic photographs showing adhesion of cells after being left in the presence of a zwitterionic polymer (polyVimC3C) are shown. Poly(VimC3C-co-C 16 Vim 1 The H-NMR chart is shown below. (a) Poly(VimC3C-co-C 16 (b) Viability of BOSC cells after cryopreservation of Poly(VimC3C-co-C 16 (c) Poly(VimC3C-co-C) K562 cells after cryopreservation. 16 Figure 1 shows the survival rate of OVMANA cells after cryopreservation using the zwitterionic polymer Poly(VimC3C-co-C 16 Figure 1 shows the toxicity of the zwitterionic polymer Poly(VimC3C-co-C) to BOSC cells. 161 shows the effect of poly(VimC3C-C16Vim) on the survival rate of K562 cells after cryopreservation. 2 shows the results of confocal microscopy after adding poly(VimC3C-C16Vim). 3 shows a schematic diagram of how the zwitterionic polymer of the present invention protects the cell membrane.

[0015] In this specification, substances having a betaine structure may be referred to as aprotic zwitterions or aprotic zwitterionic polymers, which are used synonymously with zwitterions or zwitterionic polymers.

[0016] (Biological Samples) Biological samples that can be treated with the preservative composition of the present invention include nucleic acids, proteins, organelles, cells, tissues, and individuals, with organelles, cells, and tissues being preferred. The origin of the cells that can be used is not particularly limited, and examples thereof include animal cells, insect cells, plant cells, yeast cells, and bacterial cells. Examples of animal cells include cells from humans, mice, rats, monkeys, pigs, dogs, sheep, and goats. Examples of bacteria include lactic acid bacteria, Escherichia coli, Bacillus subtilis, and cyanobacteria. The type of cell is also not particularly limited, and may be appropriately selected from the group consisting of pluripotent stem cells, tissue stem cells, somatic cells, and germ cells. Here, "pluripotent stem cells" is a general term for stem cells that have the ability to differentiate into cells of any tissue (pluripotency), and examples include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ stem cells (EG cells), and germ stem cells (GS cells). ES cells or iPS cells are preferred. Furthermore, "tissue stem cells" refer to stem cells whose differentiated cell lineages are limited to specific tissues but have the ability to differentiate into various cell types (pluripotency), such as hematopoietic stem cells in bone marrow, neural stem cells, hepatic stem cells, and skin stem cells. "Somatic cells" refer to cells that constitute multicellular organisms other than germ cells. Preferred examples include osteoclasts, fibroblasts, hepatocytes, pancreatic cells, muscle cells, bone cells, osteoblasts, chondrocytes, adipocytes, skin cells, pancreatic cells, kidney cells, lung cells, lymphocytes, erythrocytes, leukocytes, monocytes, and macrophages. "Germ cells" include gametes for sexual reproduction, i.e., eggs, oocytes, sperm, and sperm cells, as well as spores for asexual reproduction. The cells may be selected from the group consisting of sarcoma cells, established cell lines, and transformed cells. "Sarcoma" refers to cancer that develops in connective tissue cells derived from non-epithelial cells such as bone, cartilage, fat, muscle, and blood, and includes soft tissue sarcoma, malignant bone tumors, and the like. Sarcoma cells are cells derived from sarcoma. A "cell line" refers to a cultured cell that has been maintained outside the body for a long period of time, has certain stable properties, and can be subcultured semi-permanently.Examples of such cells include PC12 cells (derived from rat adrenal medulla), CHO cells (derived from Chinese hamster ovary), HEK293 cells (derived from human embryonic kidney), HL-60 cells (derived from human white blood cells), and HeLa cells (derived from human cervical carcinoma). A "transformed cell" refers to a cell whose genetic properties have been altered by introducing nucleic acid (e.g., DNA) from outside the cell. Transformation of animal cells, plant cells, and bacteria is carried out using conventionally known methods. Furthermore, when culturing ES cells or iPS cells, the culture may optionally contain feeder cells, which are used as an auxiliary agent to create an environment necessary for cell proliferation and differentiation. Examples of feeder cells include mouse fibroblasts. These feeder cells can be treated in advance with gamma irradiation or antibiotics to prevent proliferation.

[0017] (Zwitterionic Polymer) One embodiment of the zwitterionic polymer of the present invention is a zwitterionic polymer represented by the following general formula (1) or a labeled version thereof.

[0018]

[0019] (In the formula, X 1 and X 2 may be the same or different and represent a carbon atom or a nitrogen atom; Y 1 and Y 2 may be the same or different, -COO - , -SO3 - , -OP=O(H)O - , -OP=O(CH3)O - and -OP=O(OR 1 ) O -Z represents a hydrogen atom, an aromatic hydrocarbon group having 6 to 10 carbon atoms which may be substituted with an alkyl group, a 5- to 6-membered aromatic heterocyclic group which may be substituted with an alkyl group, a nitrogen-containing heterocyclic ammonium salt which may be substituted with an alkyl group, a tetraalkylammonium salt, a tetraphenylphosphonium salt, a tetraalkylphosphonium salt, a trialkylsulfonium salt, or a straight-chain or branched alkyl group having 1 to 22 carbon atoms which may have 1 to 3 oxygen atoms in the molecular chain; e and f each represent an integer of 0 or 1; l, m, and n each represent a number indicating the content ratio of each repeating unit, and are numbers which satisfy the conditions 0<l≦1, 0≦m<1, 0≦n<1, and l+m+n=1; p, q, r, s, and t each represent an integer of 0 to 6.

[0020] The zwitterionic polymer of the present invention has aprotic zwitterions, i.e., cationic and anionic moieties, in the side chains of at least l repeating units. The zwitterionic structure is represented by X in general formula (1). 1 and a heterocyclic cation containing a nitrogen atom (N) and Y 1 In addition, the side chain of m repeating units is formed with an anion of X 2 and a heterocyclic cation containing a nitrogen atom (N) and Y 2 The polymer of the present invention may have a zwitterionic structure formed by an anion of the formula (I). In the present invention, the term "zwitterionic structure" refers to a compound (internal salt) that has a positive charge and a negative charge at non-adjacent positions within the same molecule, no dissociable hydrogen atoms are bonded to the positively charged atom, and the molecule as a whole has no charge. Thus, by having zwitterions in the side chains of the repeating units of the polymer of the present invention, it is believed that the polymer exhibits a protective effect on cell membranes and functions as a cryopreservation agent. Furthermore, by adding one or more water-soluble compounds selected from electrolytes, betaines, zwitterions, alcohols, polyhydric alcohols, and sugars, the polymer of the present invention changes from an associated state due to the zwitterions in the side chains to a dispersed state, and is therefore believed to act on cell membranes and exhibit a cryoprotective effect.

[0021] In general formula (1), l, m, and n are numbers that respectively indicate the content ratio of each repeating unit, and are numbers that satisfy the following conditions: 0<l≦1, 0≦m<1, 0≦n<1, and l+m+n=1. When m=n=0, general formula (1) represents a homopolymer having a zwitterionic functional group in the side chain. When m is a number greater than 0 and n=0, general formula (1) represents a random or block copolymer having two types of zwitterionic structures. When m=0 and n is a number greater than 0, general formula (1) represents a random or block copolymer having a repeating unit having a zwitterionic functional group in the side chain and a repeating unit having Z in the side chain. When m and n are numbers greater than 0, general formula (1) represents a ternary random or block copolymer.

[0022] p, q, and t each independently represent an integer of 0 to 6. Specifically, this moiety is a single bond or a linear alkylene group having 1 to 6 carbon atoms, and a single bond, a methylene group, an ethylene group, a trimethylene group, or a tetramethylene group is preferred. Furthermore, from the viewpoint of cryoprotection of biological samples, this moiety is more preferably a single bond, a methylene group, or an ethylene group, and even more preferably a single bond or a methylene group. p, q, and t may be the same or different.

[0023] Each of r and s independently represents an integer of 0 to 6, preferably an integer of 1 to 6. This moiety is preferably a linear alkylene group having 1 to 6 carbon atoms, specifically, a methylene group, ethylene group, trimethylene group, tetramethylene group, pentamethylene group, or hexamethylene group. r and s may be the same or different.

[0024] In general formula (1), X 1 and X 2 may be the same or different and represent a carbon atom or a nitrogen atom. 1 and heterocyclic cations containing a nitrogen atom (N), such as imidazolium cations (X 1 = N, e = 0), pyridinium cation (X 1 = C, e = 1), pyrrolinium cation (X 1 = C, e = 0), pyrazinium cation (X 1Among these, imidazolium cations (X = N, e = 1) are mentioned. 1 = N, e = 0), pyridinium cation (X 1 In the general formula (1), X is preferably C, and e is preferably 1. 2 and heterocyclic cations containing a nitrogen atom (N), such as imidazolium cations (X 2 = N, f = 0), pyridinium cation (X 2 = C, f = 1), pyrrolinium cation (X 2 = C, f = 0), pyrazinium cation (X 2 Among these, imidazolium cations (X = N, f = 1) are mentioned. 2 = N, f = 0), pyridinium cation (X 2 = C, f = 1) are preferred.

[0025] Y 1 and Y 2 may be the same or different, -COO - , -SO3 - , -OP=O(H)O - , -OP=O(CH3)O - and -OP=O(OR 1 ) O - represents an anion selected from 1 and Y 2 As for -COO - , -SO3 - and -OP=O(OH)O - Preferably, the anion is selected from the group consisting of -COO - and -SO3 - More preferred are anions selected from -COO - is more preferable.

[0026] Z represents a hydrogen atom, an aromatic hydrocarbon group having 6 to 10 carbon atoms which may be substituted with an alkyl group, a 5- to 6-membered aromatic heterocyclic group which may be substituted with an alkyl group, a nitrogen-containing heterocyclic ammonium salt which may be substituted with an alkyl group, a tetraalkylammonium salt, a tetraphenylphosphonium salt, a tetraalkylphosphonium salt, a trialkylsulfonium salt, or a linear or branched alkyl group having 1 to 22 carbon atoms which may have 1 to 3 oxygen atoms in the molecular chain. Specifically, examples thereof include a hydrogen atom, a phenyl group, a naphthyl group, a C1-22 alkyl-substituted phenyl group, a C1-22 alkyl-substituted naphthyl group, an imidazolyl group, a triazolyl group, a pyridyl group, a pyrrolyl group, a pyrazinyl group, a furyl group, a thienyl group, an oxazolyl group, a thiazolyl group, pyridinium chloride, a C1-C22 alkylpyridinium chloride, imidazolinium chloride, a C1-C22 alkylimidazolinium chloride, pyridinium bromide, a C1-C22 alkylpyridinium bromide, imidazolinium bromide, a C1-C22 alkylimidazolinium bromide, pyrrolidinium chloride, a C1-C22 alkylpyridinium bromide, a C1-C22 alkylimidazolinium bromide, a C1-C22 alkylimidazolinium bromide, a C1-C22 alkylimidazolinium bromide, a C1-C22 alkylimidazolinium bromide, a C1-C22 alkylimidazolinium chloride, a C1-C22 alkylpyridinium bromide, a C1-C22 alkylimidazolinium bromide, a C1-C22 alkylimidazolinium bromide, a C1-C22 alkylimidazolinium bromide, a C1-C22 alkylimidazolinium chloride ... Examples of such alkyl groups include C22 alkylpyrrolidinium chloride, pyrrolidinium bromide, C1 to C22 alkylpyrrolidinium bromide, tetramethylammonium chloride, C2-22 alkyltrimethylammonium chloride, tetramethylammonium bromide, C2-22 alkyltrimethylammonium bromide, tetraphenylphosphonium chloride, tetraphenylphosphonium bromide, trimethylsulfonium chloride, trimethylsulfonium bromide, C1-22 linear or branched alkyl groups, ethoxyethyl groups, ethoxyethoxyethyl groups, and ethoxyethoxyethoxyethyl groups.Preferred examples of Z include a 5- to 6-membered aromatic heterocyclic group which may be substituted with an alkyl group or a nitrogen-containing heterocyclic ammonium salt which may be substituted with an alkyl group, and more preferred examples include an imidazolyl group, a triazolyl group, a pyridyl group, a pyrrolyl group, a pyrazinyl group, a furyl group, a thienyl group, an oxazolyl group, a thiazolyl group, pyridinium chloride, C1 to C22 alkylpyridinium chloride, imidazolinium chloride, C1 to C22 alkylimidazolinium chloride, pyridinium bromide, C1 to C22 alkylpyridinium bromide, imidazolinium bromide, C1 to C22 alkylimidazolinium bromide, pyrrolidinium chloride, C1 to C22 alkylpyrrolidinium chloride, pyrrolidinium bromide, and C1 to C22 alkylpyrrolidinium bromide. More preferred are an imidazolyl group, a pyridyl group, pyridinium chloride, C1 to C22 alkylpyridinium chloride, imidazolinium chloride, C1 to C22 alkylimidazolinium chloride, pyridinium bromide, C1 to C22 alkylpyridinium bromide, imidazolinium bromide, and C1 to C22 alkylimidazolinium bromide.

[0027] Labels of the zwitterionic polymer of general formula (1) include radioisotope labels, enzyme labels, chemiluminescent labels, and fluorescent labels. Among these, chemiluminescent labels and fluorescent labels are preferred. Fluorescent labels include fluorescein, cyanine, bodipy, dansyl, pyranine, coumarin, carbopyronin, and phycocyanin. Labeling of zwitterionic polymers using these labels can be achieved by binding the label to the side chain of the zwitterionic polymer or by copolymerizing a monomer having a label bound to its side chain. For example, a fluorescein-labeled polymer can be obtained by copolymerizing one or more monomers constituting the polymer represented by general formula (1) with fluorescein acrylate.

[0028] The molecular weight of the polymer compound used in the present invention must be determined based on the main chain structure, side chain structure, and overall ratio, etc., and known measurement methods that reflect the respective characteristics can be used. Furthermore, in the case of zwitterionic polymers, it is known that the main chain conformation changes depending on the type of zwitterion, pH, and the concentration of electrolytes such as NaCl. The main chain structure of the zwitterionic polymer of the present invention can be star-shaped, comb-shaped, crosslinked, or other structures, and any structure can be used for the purposes of the present invention, but the main chain conformation varies depending on the type of side chain. In the case of polymers with linear main chains, the dispersibility in water can be improved by expanding the polymer through the addition of electrolytes such as NaCl. Furthermore, the molecular weight of the zwitterionic polymer of the present invention is preferably within a certain range, since the zwitterionic structure present in the side chains affects the structure of cell membranes, cell permeability, extracellular ice crystal formation, and the solubility of poorly soluble substances. When the molecular chains are expanded by an electrolyte, the polymer preferably has an Mw of 10,000 or more and an Mn of 5,000 or more, calculated as polyethylene oxide, and more preferably an Mw of 10,000 to 2,000,000 and an Mn of 5,000 to 1,500,000. Furthermore, the molecular weight distribution of the polymer is also taken into consideration. When used as a cryopreservation agent for biological samples, the zwitterionic polymer of the present invention preferably does not permeate cell membranes, since this promotes the formation of ice crystals outside the cells.

[0029] The zwitterionic polymer of the present invention has excellent cryopreservation effects. The polymer of the present invention achieves this cryopreservation effect through a mechanism different from that of DMSO, which is conventionally used for cryopreservation. One possible mechanism is that DMSO is thought to be cell-permeable, while the zwitterionic polymer of the present invention does not enter cells and instead forms a matrix outside the cell, accumulating around the cell membrane outside the cell. One possible reason for this is that the zwitterionic molecule has a betaine structure, which gives it an electric charge and prevents it from penetrating the cell membrane as a zwitterionic polymer. Furthermore, the polymer side chain may contain a hydrophobic functional group with high affinity for cell membranes. If this functional group is inserted into the cell membrane from the outside of the cell, the hydrophilic polymer may be tethered to the cell membrane outside the cell, resulting in the polymer accumulating around the cell membrane outside the cell. Examples of functional groups that contribute to this accumulation include an alkylene group between the cation and anion in general formula (1). It is also preferable that Z has a C1-C22 alkyl group, or preferably a C3-C18 alkyl group. The ratio of such substituents to the entire polymer is preferably 0.001 to 10 mol %, more preferably 0.01 to 1 mol %, and even more preferably 0.1 to 0.5 mol %.

[0030] (Method for Producing Zwitterionic Polymers) The zwitterionic polymers of the present invention use α-olefin monomers corresponding to the repeating units described above. The polymers can be produced by radical polymerization, anionic polymerization, cationic polymerization, or the like, and these methods can be appropriately selected depending on the characteristics of the monomers and the purpose and properties of the polymer to be produced. When only one α-olefin having a betaine structure is used in the polymerization reaction, a homopolymer of a zwitterionic polymer having the same side chain (in general formula (1), l = 1, m = n = 0, or m = 1, l = n = 0) can be produced. Furthermore, when two types of α-olefins having betaine structures are used in the polymerization reaction, a copolymer of a zwitterionic polymer having two types of betaine structure side chains (0 < l < 1, 0 < m < 1, n = 0) can be produced. When one type of α-olefin having a betaine structure is polymerized with a general α-olefin, a zwitterionic polymer of polyolefin having a betaine structure in the side chain (l=0, 0<m<1, 0<n<1, or 0<l<1, m=0, 0<n<1) can be produced. Furthermore, when a zwitterionic polymer of polyolefin having a betaine structure in the side chain (l=0, 0<m<1, 0<n<1, or 0<l<1, m=0, 0<n<1) is produced, the (CH)Y 1 and (CH)Y 2 For a polymer having a structure that does not have Y at a nitrogen atom in a side chain, 1 and Y 2 Alternatively, the compound may be produced by introducing an anionic group into the (CH)Y group in the general formula (1) to cationize the nitrogen atom and subsequently construct a betaine structure in the side chain (also referred to as "post-modification"). 1 and (CH)Y 2 As the polymer having a structure not having (CH)Y in the general formula (1), a known polymer may be used. 1 and (CH)Y 2Polymers without a structure having the above structure may also be produced by polymerization. The zwitterionic polymer of the present invention can be produced by radical polymerization at high temperature and high pressure, but can also be produced at normal pressure if an appropriate polymerization initiator is used. Examples of polymerization initiators that can be used include azo compounds such as 2,2'-azobisisobutyronitrile and peroxides such as benzoyl peroxide. The polymerization initiator is used in a proportion of 0.001 mol % or more, preferably 0.01 mol %, more preferably 0.1 mol % or more, and 10 mol % or less, preferably 5 mol % or less, and more preferably 1 mol % or less, relative to the α-olefin having a betaine structure. In addition to the amount of polymerization initiator used, the molecular weight of the resulting polymer can be controlled by the nature and presence of a solvent, temperature, pressure, and other factors. Furthermore, methods for introducing anionic groups that cationize nitrogen atoms and form a betaine structure include reacting a polymer having a nitrogen atom in the side chain with a halogenated fatty acid ester, alkyl sultone, or the like. Examples of polymers having a nitrogen atom in the side chain include polypyridine and polyimidazoline.

[0031] As shown in the Examples below, although the zwitterionic polymers of the present invention are water-soluble, their aqueous solutions may have low osmotic pressures due to their large molecular weights. However, by adding a compound that contributes to osmotic pressure to the aqueous zwitterionic polymer solution of the present invention, the osmotic pressure is increased, thereby significantly improving the survival rate of biological samples after cryopreservation.

[0032] When the zwitterionic polymer of the present invention is used in a dispersion containing a biological sample such as cells, it is preferable to add one or more water-soluble compounds selected from electrolytes, betaine, zwitterions, alcohols, polyhydric alcohols, and sugars to the dispersion to adjust the osmotic pressure. Adjusting the osmotic pressure can further improve the ability to maintain the survival and function of the biological sample when the biological sample is cryopreserved. Therefore, a composition containing the zwitterionic polymer of the present invention is useful as a preservative composition for biological samples, particularly as a cryopreservative composition for biological samples. Furthermore, the preservative composition for biological samples of the present invention may be in the form of a culture medium composition for biological samples. Of these, electrolytes are more preferable for adjusting the osmotic pressure from the viewpoint of the cryopreservation effect of biological samples. Furthermore, considering toxicity to biological samples, sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, sodium bicarbonate, and potassium bicarbonate are preferred electrolytes.

[0033] The effects of the present invention can be more pronounced by adding one or more water-soluble compounds selected from electrolytes, betaine, zwitterions, alcohols, polyhydric alcohols, and sugars to aqueous solutions, culture media, cryopreservation solutions, etc. containing the zwitterionic polymers of the present invention. These water-soluble compounds can change the conformation of the entire polymer by acting on the zwitterionic side chains of the zwitterionic polymer. When water-soluble compounds such as electrolytes that act on the side chains of zwitterionic polymers are incorporated into the zwitterionic polymer by forming salts with the side chains, the concentration that contributes to increasing osmotic pressure may not be achieved. Therefore, the type and amount of the water-soluble compound added must be considered in relation to the structure of the zwitterionic polymer. Here, an electrolyte is a substance that dissociates into cations and anions when dissolved in a solvent. Preferred cations include sodium ions, potassium ions, calcium ions, magnesium ions, etc., and preferred anions include chloride ions, phosphate ions, bicarbonate ions, etc. Therefore, a preferred electrolyte is a compound that combines a cation selected from sodium ions, potassium ions, calcium ions, and magnesium ions with an anion selected from chloride ions, phosphate ions, and bicarbonate ions. Specific examples include sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, sodium bicarbonate, and potassium bicarbonate. Alcohols include alcohols having 1 to 6 carbon atoms, such as methanol, ethanol, and isopropanol. Polyhydric alcohols include water-soluble polyhydric alcohols, such as ethylene glycol and propylene glycol. Sugars include monosaccharides and disaccharides, such as glucose and sucrose. Betaines include carnitine. Zwitterions include those described in WO2020 / 230721.

[0034] (Polymer Aggregation) Furthermore, particle size measurements by dynamic light scattering (DLS) of the zwitterionic polymer aqueous solution of the present invention revealed the presence of many particles in the 100-1,000 nm range. Addition of an electrolyte reduced the degree of aggregation, and in some cases aggregation was barely observed. After the addition of electrolyte, the presence of many particles near 10 nm was observed ( Figure 19 ). In aqueous solution, the zwitterionic polymer of the present invention aggregates depending on the density of the side chains. However, interaction between the electrolyte and the side chains causes the aggregated polymer to form ion pairs with the electrolyte, resulting in a conformational change in the polymer backbone, expanding the shape of the aggregated polymer and resulting in a dispersed state. The addition of an electrolyte not only increases osmotic pressure, but also contributes to the more dispersed state of the zwitterionic side chains as zwitterionic monomers, which is thought to contribute to shelf life. DLS observations indicate that the zwitterionic polymer of the present invention contributes to shelf life by becoming more dispersed in an aqueous solution containing an electrolyte. When the polymer becomes dispersed, the polymer backbone is widely dispersed to cover the biological surface, and the side chains interact with or penetrate the biological membrane, enhancing its affinity with the biological membrane. As a result, the zwitterionic polymer interacts with the biological sample being preserved, particularly the cell membrane, forming a matrix-like structure around the cells. This interaction between the zwitterionic polymer and the cell membrane also affects the relationship between cells, resulting in the observation of cell aggregation (Figure 27). Cells observed in the medium containing the zwitterionic polymer of the present invention appear to be clustered together, in contrast to the individual cells observed in PBS. This suggests that the addition of the zwitterionic polymer of the present invention may have some effect on cell adhesion. Although the magnitude of this adhesive effect is unclear, it is expected that this polymer will be useful in the future for organ fixation during surgery, such as organ transplantation, and for the production of tissues and organs using three-dimensional printers. The effect of increasing the ice melting temperature was observed by measuring the temperature at which ice melts using DSC during the temperature raising process of returning the sample to room temperature after rapid freezing.These results suggest that the zwitterionic polymer, when dispersed in electrolytes, interacts with the cell membrane, causing some structural changes that protect the cells. For example, this protective effect may prevent ice from entering the cells, maintaining cell viability. Furthermore, the extracellular protective effect of the zwitterionic polymer makes it possible to culture individual animal cells as suspension cells in a medium.

[0035] (Preservative Composition) The preservative composition for biological materials of the present invention not only functions as a cryopreservative for the biological material, but also functions as a composition for preserving and maintaining the biological material. Specific examples include cryopreservative compositions for biological materials, compositions for culturing biological materials, medium compositions for biological materials, compositions for preserving biological materials, compositions for maintaining the function of biological materials, and compositions for testing the function of biological materials. Examples of the medium for the preservative composition for biological samples of the present invention include water and mixtures of water and organic solvents. The medium selected is preferably a substance in which the electrolyte ionizes into cations and anions. Examples of organic solvents include lower alcohols such as methanol and ethanol, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF).

[0036] When the zwitterionic polymer or a labeled product thereof of the present invention is used as a preservative for a biological sample, the concentration of the zwitterionic polymer is not particularly limited, but is preferably 2% by mass to 30% by mass, more preferably 5% by mass to 30% by mass, and even more preferably 10% by mass to 20% by mass. The concentration of one or more water-soluble compounds selected from electrolytes, betaines, zwitterions, alcohols, polyhydric alcohols, and sugars is preferably a concentration that does not affect the viability of the biological sample, and is preferably 0.2% by mass to 5% by mass, more preferably 0.5% by mass to 3% by mass, and even more preferably 0.5% by mass to 2% by mass.

[0037] Furthermore, the preservative composition of the present invention may contain a cell-permeating substance as an additive, added to 100 parts by weight of a composition containing an aprotic zwitterion and water or a composition consisting of an aprotic zwitterion and water (also referred to as an aprotic zwitterion aqueous solution). The cell-permeating substance may be added in an amount of typically at least 1 part by weight, preferably 10 parts by weight or more, with an upper limit of typically 30 parts by weight or less, preferably 20 parts by weight or less, and more preferably 15 parts by weight or less, per 100 parts by weight of the aprotic zwitterion aqueous solution. In this specification, the addition of 1 part by weight of the cell-permeating substance to 100 parts by weight of the aprotic zwitterion aqueous solution may be referred to as 1% of the aprotic ion aqueous solution. Examples of such cell-permeating substances include dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propylene glycol. Such cell-permeating substances can further improve cryopreservation properties. The amount of these cell-permeating substances added may be increased or decreased relative to the amount typically used. For example, in the case of DMSO, the concentration can be 3% by mass or more and 25% by mass or less, and in the case of glycerol, the concentration can be 3% by mass or more and 25% by mass or less.

[0038] The preservative composition of the present invention can also contain components of a medium for culturing a biological sample. In this case, the preservative composition of the present invention functions as a medium composition or a culture composition. Examples of medium components for culturing a biological sample include medium components for cell culture, specifically inorganic salts, buffers, carbohydrates, vitamins, proteins, peptides, fatty acids, lipids, trace elements, serum, hormones, growth factors, signal transduction substances, antibiotics, etc. When the composition of the present invention is used as a medium composition, it is preferable that it contains these medium components.

[0039] The preservative composition of the present invention improves the cryopreservation properties of a dispersion of cells, etc., and is therefore also useful as an additive to a culture medium for cells, etc. Therefore, a composition containing the preservative of the present invention and cell culture medium components can be used as a culture medium for cryopreserving cells, etc. Here, the cell culture medium components include, in addition to the zwitterionic polymer of the present invention, electrolytes, and cell-permeating substances, nutrients (e.g., sugars for cell growth), peptides, and proteins (e.g., serum, proteins and peptides purified from serum, etc.).

[0040] The preservative composition of the present invention can improve the viability of cells thawed after freezing a biological sample dispersion, such as cells, using slow or rapid freezing. The freezing conditions for cells in the slow freezing method can be appropriately set in accordance with conventional conditions. Specifically, for example, cells can be cooled to 0 to -200°C at a cooling rate of -0.1 to -15°C / min. Rapid freezing is preferably performed at a cooling rate of -15 to -20,000°C / min and a cooling temperature of 0 to -200°C. A preferred method for thawing cryopreserved cells is to quickly transfer an ampoule containing the frozen cells to a 37°C water bath. The contents of the ampoule are transferred to a sterile tube using a pipette. Then, preheated medium supplemented with appropriate supplements is gradually added. The viable cell density is measured using trypan blue. An appropriate amount of the cell suspension is transferred to a flask and seeded at the cell density recommended in the cell line's data sheet.

[0041] (Dissolution of Poorly Soluble Substances) In the present invention, the term "poorly soluble substance" refers to a substance that has the property of being only slightly soluble in water, and is a substance whose solubility in water (25°C) is 1% by weight or less, preferably 0.5% by weight or less, and particularly preferably 0.1% by weight or less. Such poorly soluble substances include, for example, substances that are active ingredients of pharmaceuticals, veterinary drugs, quasi-drugs, cosmetics, and agricultural chemicals (including candidate substances that may become active ingredients), food additives, biological substances, and plant-derived substances, and include low molecular weight substances, and oligomers and polymers such as oligopeptides, polypeptides, polysaccharides, DNA, and RNA. "Slightly soluble pharmaceuticals" refer to drugs that are "slightly soluble," "slightly soluble," "extremely soluble," or "almost insoluble" as defined in the Japanese Pharmacopoeia, and specifically include antitumor agents, antibiotics, antihyperlipidemic agents, antibacterial agents, allergic disease treatments, hypertension treatments, arteriosclerosis treatments, blood circulation promoters, hormones, fat-soluble vitamins, diabetes treatments, antiandrogens, cardiotonic drugs, antiarrhythmic drugs, anti-inflammatory drugs, hypnotics, sedatives, tranquilizers, antiepileptic drugs, antidepressants, digestive system disease treatments, diuretics, local anesthetics, anticoagulants, antihistamines, etc. Examples of poorly soluble substances include steroids, antimuscarinics, antimycobacterial agents, immunosuppressants, antithyroid drugs, antivirals, anxiolytic sedatives, astringents, β-adrenergic receptor blockers, cardiac inotropes, contrast media, corticosteroids, cough suppressants, diagnostic agents, diagnostic imaging agents, diuretics, dopaminergic agents, lipid-regulating agents, muscle relaxants, parasympathomimetics, thyroid calcitonin, prostaglandins, radiopharmaceuticals, sex hormones, stimulants, appetite suppressants, sympathomimetics, thyroid medications, vasodilators, isoflavones, and xanthenes. More specific examples of poorly soluble substances include glycyrrhetinic acid and its salts, glycyrrhizinic acid and its salts, coumarin, ononin, liquiritin, peptides, polypeptides such as collagen, polysaccharides such as xylan, lignin, and chloramphenicol. The addition of the zwitterionic polymer of the present invention can improve the solubility of these poorly soluble substances.Furthermore, by dissolving a poorly soluble substance in a solution containing the zwitterionic polymer of the present invention and one or more water-soluble compounds selected from electrolytes, betaines, zwitterions, alcohols, polyhydric alcohols, and sugars, the solubility of the poorly soluble substance is significantly improved.

[0042] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0043] (Pure water) In the examples, water used was passed through a Sartorius ultrapure water production system. (Cells) Mouse Normal Fibroblasts (mNF) established from C57BL / 6-EGFP mice were used. Human kidney cells (BOSC) and rat glioma cells (C6 cells) were obtained from the Hirata Laboratory at Kanazawa University. Human chronic myeloid leukemia cells (K562 cells) and mouse glial-neuronal co-expressing cells (Vn1919 cells) were obtained from the Wong Laboratory at Kanazawa University. OVMANA cells were purchased from the JCRB Cell Bank at the National Institutes of Biomedical Innovation, Health and Nutrition. ( 1 H-NMR) 1 H-NMR was measured using an ECA400 (external magnetic field 400 MHz) manufactured by JEOL Ltd. (GPC) A TSKgel α-M column (Tosoh) was used as the stationary phase. Pure water was used as the mobile phase, with a flow rate of 1.0 mL / min and a measurement temperature of 40°C. Detection was performed using a refractive index detector (RID-10A), and various molecular weights were calculated in terms of polyethylene oxide. (DSC) A DSC device manufactured by Shimadzu Corporation was used. The sample was cooled to -150°C at 1°C / min, and then returned to room temperature at 5°C / min. The temperature at which the ice melted was measured during the heating process. (DLS) A DLS device manufactured by Horiba Ltd. was used to measure particle size distribution. The solution passed through a 3 μm filter was placed in a disposable cell, and the particle size was measured at a set temperature of 25°C. (Osmotic Pressure) Measurement was performed in a 10 mL standard chamber using a vapor pressure type osmometer (VAPRO 5600; Wescor, Inc., Logan, UT, USA) (room temperature: 25°C, device sensor temperature: 33°C, sample volume: 10 μL).

[0044] (Cryopreservation Test) (a) Cryopreservation of Cells In the case of adherent cells, cells to be frozen were treated with trypsin and collected by centrifugation. In the case of suspension cells, cells to be frozen were collected by centrifugation. The cells were diluted with Dulbecco's modified Eagle's medium (DMEM) and the cell concentration was measured. The DMEM solution may be referred to simply as "medium" in this specification. Subsequently, 1 mL aliquots were dispensed into 1.5 mL tubes, centrifuged, and suspended in a cryopreservation solution containing 100 μL of zwitterionic polymer solution. The cells were then frozen using a cell freezing container, Mr. Frosty®, at a cooling rate of -1°C / min and a cooling temperature of -85°C. In this example, unless otherwise noted, CultureSure (registered trademark, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used as the commercially available cryopreservation solution. After 48 hours of cryopreservation, the tubes were thawed at 37°C and used to count the viable cells. (b) Thawing of cells and counting of viable cell counts 1 mL of medium was added to a cryopreservation vial at 37°C, followed by thawing and centrifugation to remove the supernatant. The cells obtained by centrifugation were then resuspended in medium and the viable cell count was counted. (Cytotoxicity test) The cells were allowed to stand in the polymer solution at room temperature for 60 minutes, and the proportion of dead cells in the cells was measured by trypan blue staining. (Cell proliferation test) The cells were cryopreserved according to the cryopreservation test described above, and then cultured for a certain period in medium (DMEM containing 10% FBS and 1% antibiotics), and the cell proliferation count from before culture was counted.

[0045] (Example 1) (1) Production of VimC3C 15.69 g (0.167 mmol) of vinylimidazole (Tokyo Chemical Industry Co., Ltd.) was added to 100 mL of tetrahydrofuran (Fujifilm Wako Pure Chemical Industries Co., Ltd.), and 32.48 g (0.167 mmol) of 4-bromobutyric acid ethyl ester (Tokyo Chemical Industry Co., Ltd.) was added, followed by refluxing at 70°C for 24 hours. After washing with ethyl acetate, the mixture was applied to a column of anion exchange resin (Amberlite IRN 78A). The eluate was distilled off under reduced pressure to obtain VimC3C. VimC3C (DMSO) 1 The H-NMR chart is shown in Figure 1.

[0046] (2) Production of Poly(VimC3C)

[0047]

[0048] 3 g (0.014 mmol) of VimC3C was added to 10 mL of pure water, and 22.83 g (0.0014 mmol) of 2,2'-azobis(isobutyronitrile) (Tokyo Chemical Industry Co., Ltd.) was added as a polymerization initiator, followed by stirring at 80°C for 16 hours. Poly(VimC3C) was obtained by dialysis against pure water and drying under reduced pressure. Poly(VimC3C) (methanol) 1 The H-NMR spectrum is shown in Figure 2. 1 H-NMR is shown in Figure 3. The reaction was carried out by adding 0.01 to 10 mol% of the polymerization initiator in terms of the monomer ratio. In the examples of this specification, for example, a solution in which a polymer produced by adding 10 mol% of the polymerization initiator in terms of the monomer ratio as in this example was added at 10 mass% relative to the total solution is referred to as 10%10 mol% Poly(VimC3C). Unless otherwise specified regarding the amount of polymerization initiator, it is assumed that a polymer produced by adding 1 mol% of the polymerization initiator in terms of the monomer ratio is used.

[0049] Example 2: Production method of Poly(VpyC3C)

[0050]

[0051] To 20 mL of dimethyl sulfoxide (Kanto Chemical Co., Inc.), 4.95 g (0.0033 mmol) of poly(4-vinylpyridine) (Merck Co., Ltd.) was added, and 3.2 g (0.0033 mmol) of 4-bromobutyric acid ethyl ester (Tokyo Chemical Industry Co., Ltd.) was added, followed by stirring at room temperature for 24 hours. The mixture was washed with ethyl acetate and dried under reduced pressure to obtain Poly(VpyC3C). The obtained Poly(VpyC3C) (methanol-d6) 1 The H-NMR chart is shown in Figure 4. Mw = 34321, Mn = 19981, melting point (Tm) = 149°C.

[0052] Example 3: Production method of Poly(VpyC3S)

[0053]

[0054] To 10 mL of dichloromethane (Kanto Chemical Co., Inc.), 0.92 g (0.0018 mmol) of poly(4-vinylpyridine) (Merck Co., Ltd.) was added, and 1.1 g (0.0018 mmol) of 1,3-propane sultone (Tokyo Chemical Industry Co., Ltd.) was added, followed by stirring at room temperature for 24 hours. Poly(VpyC3S) was obtained by washing with ethyl acetate and drying under reduced pressure. The obtained Poly(VpyC3S) (DO, 50°C) 1 The H-NMR chart is shown in FIG.

[0055] (Example 4) (1) Production of VimCS 21.76 g (0.23 mmol) of vinylimidazole (Tokyo Chemical Industry Co., Ltd.) was added to 150 mL of tetrahydrofuran (Fujifilm Wako Pure Chemical Industries Co., Ltd.), and 28.24 g (0.23 mmol) of 1,3-propane sultone (Tokyo Chemical Industry Co., Ltd.) was added, followed by stirring at room temperature for 24 hours. VimCS was obtained by washing with ethyl acetate and drying under reduced pressure. The obtained VimCS (DO) 1 The H-NMR chart is shown in FIG.

[0056] (2) Production of Poly(VimC3S)

[0057]

[0058] To 10 mL of a 2% aqueous solution of NaCl (Nacalai Tesque, Inc.), 3 g (0.017 mmol) of VimCS was added, followed by 27.42 g (0.00017 mmol) of 2,2'-azobis(isobutyronitrile) (Tokyo Chemical Industry Co., Ltd.), and the mixture was stirred at 80°C for 16 hours. Poly(VimCS) was obtained by reprecipitation with pure water and drying under reduced pressure. The obtained Poly(VimCS)(DO) 1 The H-NMR chart is shown in FIG.

[0059] (Example 5-1) Production of Poly(VpyC3C)50

[0060]

[0061] Poly(4-vinylpyridine) (Mw: 160,000, Merck) (5.53 g, 0.01 mmol) was dissolved in dichloromethane, and 4-bromobutyric acid ethyl ester (2.31 g, 0.01 mmol, Tokyo Chemical Industry Co., Ltd.) was added, followed by refluxing at 50°C for 17 hours. After refluxing, the solution was washed with ethyl acetate, purified using an ion exchange resin (Amberlite IRN78 Hydroide DSM), and then dialyzed using Spwctra / por 7 Dialysis Membrane Pre-treated RC Tubing MWCO 1 kD to obtain 4.88 g of Poly(VpyCC)50. 1 The H-NMR (CHOH-d) chart is shown in Figure 8. 1 H-NMR signals showed m:n=1:1. Mw=249086, Mn=204914.

[0062] (Example 5-2) Poly(VpyC3C) 40 3.59 g of Poly(VpyC3C) 40 was obtained by the same procedure as in Example 5-1, except that 4-bromobutyric acid ethyl ester (2.31 g, 0.01 mmol, Tokyo Chemical Industry Co., Ltd.) was used instead of 4-bromobutyric acid ethyl ester (1.91 g, 0.008 mmol, Tokyo Chemical Industry Co., Ltd.). 1 The H-NMR (CHOH-d) chart is shown in Figure 9. From the NMR signals, m:n=3:2.

[0063] (Example 5-3) Poly(VpyC3C)30 2.79 g of Poly(VpyC3C)30 was obtained by the same procedure as in Example 5-1, except that 4-bromobutyric acid ethyl ester (2.31 g, 0.01 mmol, Tokyo Chemical Industry Co., Ltd.) was used instead of 4-bromobutyric acid ethyl ester (0.97 g, 0.005 mmol, Tokyo Chemical Industry Co., Ltd.). 1 The H-NMR (CHOH-d) chart is shown in Figure 10. The NMR signals indicated that m:n = 3.3:1.

[0064] (Example 5-4) Poly(VpyC3C)20 2.32 g of Poly(VpyC3C)20 was obtained by the same procedure as in Example 5-1, except that 4-bromobutyric acid ethyl ester (2.31 g, 0.01 mmol, Tokyo Chemical Industry Co., Ltd.) was used instead of 4-bromobutyric acid ethyl ester (0.77 g, 0.003 mmol, Tokyo Chemical Industry Co., Ltd.). 1 The H-NMR (CHOH-d) chart is shown in FIG. 1 From the H-NMR signals, m:n=4:1.

[0065] (Example 5-5) Poly(VpyC3C)10 1.95 g of Poly(VpyC3C)20 was obtained by the same procedure as in Example 5-1, except that 4-bromobutyric acid ethyl ester (2.31 g, 0.01 mmol, Tokyo Chemical Industry Co., Ltd.) was used instead of 4-bromobutyric acid ethyl ester (0.48 g, 0.001 mmol, Tokyo Chemical Industry Co., Ltd.). 1 The H-NMR (CHOH-d) chart is shown in FIG. 1 From the H-NMR signals, m:n=9:1.

[0066] Example 6-1: Preparation of Poly(VpyC3S)40

[0067]

[0068] Poly(4-vinylpyridine) (Mw: 160000, Merck Ltd.) (1.97 g, 0.04 mmol) was dissolved in dichloromethane in a 2% NaCl solution of water:methanol (10 mL:10 mL), and 1,3-propane sultone (0.84 g, 0.006 mmol, Tokyo Chemical Industry Co., Ltd.) was added and reacted at 25°C for 24 hours. The reaction solution was treated with a large amount of water to cause reprecipitation. The precipitate was freeze-dried to obtain Poly(VpyCS)40 (2.05 g). 1The H-NMR (methanol-NaCl) chart is shown in Figure 13. The signal of the proton of the methylene group bonded to pyridine (signal 13 in Figure 13) overlapped with the signal of water and could not be confirmed, so it was heated to 50°C and measured, and a signal appeared at 4.9 ppm. 1 From the H-NMR signals, m:n=3:2.

[0069] Example 6-2 Poly(VpyCS) 15 was obtained by the same reaction as in Example 6-1, except that 1,3-propane sultone (0.23 g, 0.002 mmol, Tokyo Chemical Industry Co., Ltd.) was used instead of 1,3-propane sultone (0.84 g, 0.006 mmol, Tokyo Chemical Industry Co., Ltd.) in Example 6-1. 1 From the H-NMR (CHOH—NaCl) signals, m:n was 17:3.

[0070] Example 7: Production of Poly(VimC3C-co-C8Vim)

[0071]

[0072] 4 mL of methanol was added to 1 g of VimCC and [C3Vim]Cl (charge ratio m:n = 1:1) (1.375 M) to dissolve these monomers. 10 mol% of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride was added, and the mixture was ultrasonicated for 180 minutes, followed by nitrogen substitution and stirring in a 60°C oil bath for 17 hours. 100 mL of DMSO (poor solvent) was cooled with cold water and stirred, and the polymerization reaction solution dissolved in methanol was added dropwise thereto. Stirring continued until the polymerization reaction solution solidified in the poor solvent. The mixture was filtered under reduced pressure using a membrane filter, and finally, the DMSO and monomers in the copolymer were washed away with acetone, and the copolymer was recovered from the membrane filter. The recovered copolymer was stirred with a large amount of chloroform to remove DMSO. The resulting Poly(VimCC-co-C8Vim) 1 The H-NMR (CDOD) chart is shown in FIG.

[0073] (Example 8)

[0074] 1-Vinyl-imidazole was refluxed with pentadecane chloride at 70°C without solvent to obtain [C16Vim]Cl.

[0075]

[0076] (A) Preparation of Poly(VimCC-co-C16Vim)-1.0 4 mL of methanol was added to 1 g of VimCC and [C16Vim]Cl (charge ratio m:n = 1:1) (1.375 M) to dissolve these monomers. 10 mol% of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride was added, and the mixture was sonicated for 180 minutes, then purged with nitrogen, and refluxed in an oil bath at 75°C for 17 hours. This was followed by dialysis for three days to purify the product, yielding poly(ZI-C16) (abbreviated as "(A)C16P"). 1 The proportion of C16Vim units in the purified polymer determined by H-NMR was 0.34 mol %, and the molecular weight was 84 kDa. The results are shown in Table 5.

[0077] (ii) Production of Poly(VimCC-co-C16Vim)-0.5 Poly(ZI-C16) (abbreviated as (ii)C16P) was obtained in the same manner as in the synthesis of (i)C16P, except that the feed ratio 1 was changed to 1:0.5. 1 The proportion of C16Vim units in the purified polymer determined by H-NMR was 0.21 mol %, and the molecular weight was 374 kDa. The results are shown in Table 5.

[0078] (C) Production of Poly(VimCC-co-C16Vim)-0.1 Poly(ZI-C16) (abbreviated as (C)C16P) was obtained in the same manner as in the synthesis of (A)C16P, except that the feed ratio 1 was changed to 1:0.1. 1 The proportion of C16Vim units in the purified polymer determined by H-NMR was 0.05 mol %, and the molecular weight was 178 kDa. The results are shown in Table 5. 1 The H-NMR chart is shown in FIG.

[0079] Example 9: VimC3C (zwitterionic monomer) and Poly(VimC3C) (zwitterionic polymer) prepared in Example 1 were dissolved in water to prepare 10% by mass preservation solutions, and viability was measured using BOSC cells. The osmotic pressure of the polymers and cell viability after cryopreservation were also measured, and the results are shown in Table 1. The zwitterionic polymer aqueous solution exhibited poorer viability than the zwitterionic monomer aqueous solution. Cells preserved in the zwitterionic polymer aqueous solution were observed to absorb water, swell, and die. This was thought to be due to the fact that when cells were cryopreserved in the polymer aqueous solution alone, the increased molecular weight resulted in a hypotonic solution with low osmotic pressure, even at the same weight. Therefore, to adjust the osmotic pressure, NaCl was added to the polymer aqueous solution and the osmotic pressure was measured. The results are shown in Table 2. It was found that the addition of 2% NaCl achieved an osmotic pressure comparable to that of 10% by mass zwitterionic monomer. As shown in Example 10 below, high cell viability was achieved by using a medium with improved osmotic pressure.

[0080]

[0081]

[0082] Example 10 To investigate the relationship between polymer concentration and cell viability after cryopreservation, the viability of cells (BOSC, mNF) after cryopreservation was measured when Poly(VimC3C) polymer DMEM solution (1 mass%, 5 mass%, 10 mass%, 20 mass%, 30 mass%) was used as the cryopreservation solution. The results are shown in Figure 15. As the polymer concentration increased, the cell viability increased, reaching a maximum at 10 to 20 mass%. This indicates that there is an optimal value for the polymer concentration.

[0083] Example 11: To investigate the effect of NaCl addition to the polymer on the survival rate of cells after cryopreservation, solutions containing 10% Poly(VimC3C) polymer aqueous solution with NaCl (0.1% by mass, 0.5% by mass, 0.8% by mass, 1% by mass, and 2% by mass) were used as cell cryopreservation solutions, and the survival rate of cells (BOSC, mNF) after cryopreservation was measured. The results are shown in Figure 16. The optimal NaCl concentration was around 1% by mass, indicating that an isotonic solution is most effective for cells. This suggests the possibility of using the solution as both a culture medium and a preservation medium for cells.

[0084] Example 12: To investigate the type of solute used to adjust osmotic pressure, sucrose (6% aqueous solution), trimethylglycine (2% aqueous solution), VimC3C (3% aqueous solution of monomer), and [C2mim]OAc (3% aqueous solution of monomer) were prepared and added in predetermined amounts to a 10% Poly(VimC3C) solution to prepare cryopreservation solutions, and the viability of cells (BOSC, mNF) after cryopreservation was measured. The relationship between osmotic pressure and cell viability is shown in Table 3 and Figure 17. The cryopreservation solutions containing the zwitterionic monomer had low osmotic pressure, possibly due to the small amount added, and were unable to increase cell viability.

[0085]

[0086] Example 13 To examine the cytotoxicity of the polymer, cells (BOSCs) were placed in a medium containing the polymer. The results are shown in Figure 18. The zwitterionic polymer-containing solution of the present invention was found to have lower cytotoxicity than solutions containing other substances. This suggests that cells can be cultured in this polymer-containing medium. Furthermore, the cell death rate was lower than in a medium without the polymer, suggesting the possibility of culturing adherent cells in a suspension state. The low cell death rate in the polymer solution is thought to be due to the cells being covered by a polymer matrix dispersed by the electrolyte, or the cells adhering to each other via the matrix, preventing cell death during freezing and thawing. These results also suggest that the matrix formed by the polymer dispersed by the electrolyte may act as a cell adhesive.

[0087] (Example 14) To investigate the effect of adding an electrolyte to a polymer on the formation of ice crystals in the solution, the polymer solution was measured by DSC. The DSC of the polymer NaCl solution and the ice crystal ratio after frozen storage are shown in Table 4. As a result, it was found that the polymer NaCl solution interacted with water.

[0088]

[0089] Example 15: To investigate the effect of adding electrolytes to a polymer on aggregation, a polymer solution was measured by DLS. Figure 19 shows the DLS chart of a polymer NaCl solution. Figure 19 reveals that the peak shifts to the left as the electrolyte concentration increases, indicating that the addition of electrolytes prevents polymer aggregation. From the results in Table 4 and Figure 19, it is believed that the zwitterionic polymer of the present invention prevents cells from dying due to freezing and thawing by dispersing the polymer in the electrolyte and interacting with the cell membrane. It is believed that cell death during freezing and thawing is prevented by covering the cells with a matrix of the polymer dispersed in the electrolyte, or by adhering the cells to each other via the matrix.

[0090] Example 16 To investigate the effect of different polymer molecular weights on cell survival rates after cryopreservation, we measured the survival rates of cells (BOSC, mNF) after cryopreservation when 10% Poly(VimC3C) solutions with different molecular weights were used as cryopreservation solutions. Figure 20 shows the GPC results for the polymers, and Figure 21 shows the relationship between the polymer and cell survival rates. As shown in Figure 20, increasing the amount of polymerization initiator tended to decrease the molecular weight of the polymer. Furthermore, as shown in Figure 21, when the amount of polymerization initiator was between 0.1 mol% and 10 mol% relative to the monomer, the survival rate of cells after cryopreservation was not affected, and the polymer could be used as a cryopreservation solution as long as its molecular weight was at least in the range of Mw 100,000 to 250,000 and Mn 50,000 to 200,000.

[0091] Example 17 To examine the effect of using a polymer solution as a cryopreservation solution on the survival rate of other cells after cryopreservation, the survival rate after cryopreservation was measured using three types of cells (K562 cells, C6 cells, and OVMANA cells). The results are shown in Figures 22, 23, and 24. It was found that when the polymer of the present invention is used as a cryopreservation solution, a cell survival rate after cryopreservation comparable to that of commercially available products was obtained. In particular, K562 cells and OVMANA cells, which are known to be sensitive to cryopreservation, achieved higher survival rates than when commercially available cryopreservation solutions were used.

[0092] Example 18 To examine the effect of the polymer solution on cell proliferation rate, cells (C6 cells and OVMANA cells) were cryopreserved, and then the C6 cells were cultured for 48 hours and the OVMANA cells for 18 days, and the cell proliferation rate was measured. The results are shown in Figures 25 and 26. Proliferation was confirmed for cells cryopreserved using a solution containing the zwitterionic polymer of the present invention. A higher cell number was confirmed for OVMANA cells than for the commercially available product. C6 cells proliferated, although the results were slightly inferior to those for the commercially available product.

[0093] Example 19 To observe cells in polymer solution, cells were placed in PBS or 10% Poly(VimC3C) 1% NaCl solution at room temperature for 60 minutes. Microscopic observation results are shown in Figure 27. As a result, (a) cells in PBS were evenly dispersed, whereas (b) cells in polymer solution were adhered to each other. This suggests that the polymer forms a matrix around the cells.

[0094] Example 20: The cryopreservation effect of poly(VimC3C-C16Vim) synthesized in Example 8 was investigated. Poly(ZI-C16) polymers with molar contents of C16 monomer-derived units of 0.05 mol% (Example 8(c)), 0.21 mol% (Example 8(b)), and 0.34 mol% (Example 8(a)) were cryopreserved at 10 wt %, and the viability of BOSC cells, K562 cells, and OVMANA cells was investigated. The polymer synthesized in Example 1 was used as the polymer with a 0 mol % C16 monomer-derived unit content. The results are shown in Figure 29. A C16-derived unit content of 0.05 mol % was effective in all cell lines.

[0095]

[0096] Example 21 The toxicity of poly(ZI-C16) synthesized in Example 8 to BOSC cells was investigated. Culture media containing 10 wt % of poly(ZI-C16) polymers with molar contents of C16 monomer-derived units of 0.05 mol % (Example 8(c)), 0.21 mol % (Example 8(b)), and 0.34 mol % (Example 8(a)) were used. 6 The BOSC cells were incubated at 37°C for 1 hour, and then the viable cells were counted to determine the survival rate. The polymer synthesized in Example 1 was used as the polymer with a 0 mol% content of units derived from C16 monomer. The results are shown in Figure 30. No C16 content-dependent toxicity was observed for (a) to (c).

[0097] (Example 22) The effect of salt concentration on the cryopreservation effect of poly(ZI-C16) synthesized in Example 8 was investigated. 10 wt% media (Example 8(c)) were prepared with NaCl concentrations of 0.5 wt%, 1 wt%, and 2 wt%, and K562 cells were cryopreserved and the preservation rate was investigated. The results are shown in Figure 31. The solution with 1 wt% NaCl was the best.

[0098] Example 23 Synthesis of Fluorescent Label Compound Poly(ZI-C16-Fluorescein)

[0099]

[0100] VimC3C, [C16Vim]Cl, and fluorescein o-acrylate were added to MeOH in a molar ratio of 1:1:1, and AIBN was added. The mixture was reacted at 75°C for 17 hours. After cooling, the mixture was dialyzed for 3 days to obtain poly(ZI-C16-Fluorescein) as a fluorescently labeled compound.

[0101] (Discussion of Mechanism) A synthesized fluorescent label was added to the culture medium of BOSC cells. After 20 minutes, the cells were washed with PBS and observed under a confocal microscope. The results are shown in Figure 32. Figure 32 reveals that poly(VimC3C-C16Vim) accumulates around the cells, particularly at the cell membrane. This suggests that poly(VimC3C-C16Vim) forms an extracellular matrix, particularly on the cell membrane, preventing the influx of ice crystals from outside the cell. Furthermore, it was possible to speculate that the alkyl side chains of poly(VimC3C-C16Vim) are inserted into the cell membrane and tethered to the outside of the membrane, thereby protecting the cell membrane and preventing the influx of ice crystals. This is illustrated schematically in Figure 33.

Claims

1. A zwitterionic polymer represented by the following general formula (1) or a labeled product thereof. 【Chemistry 1】 (In the formula, X 1 and X 2 These may be the same or different, and represent a carbon atom or a nitrogen atom; Y 1 and Y 2 may be the same or different, -COO - , -SO 3 - , -OP=O(H)O - , -OP=O(CH 3 )O - and -OP=O(OR 1 )O - represents an anion selected from; Z represents a hydrogen atom, a C6-C10 aromatic hydrocarbon group which may be substituted with an alkyl group, a C5-C6 membered aromatic heterocyclic group which may be substituted with an alkyl group, a nitrogen-containing heterocyclic ammonium salt which may be substituted with an alkyl group, a tetraalkylammonium salt, a tetraphenylphosphonium salt, a tetraalkylphosphonium salt, a trialkylsulfonium salt, or a C1-C22 linear or branched alkyl group which may have 1-3 oxygen atoms in its molecular chain; e and f represent integers 0 or 1, respectively; l, m, and n are numbers that represent the content ratio of each repeating unit, respectively, and satisfy the conditions 0 < l ≤ 1, 0 ≤ m < 1, 0 ≤ n < 1, and l + m + n = 1; (p, q, r, s, and t each represent integers from 0 to 6.)

2. Y 1 and Y 2 However, they may be the same or different, -COO - and -SO 3 - The zwitterionic polymer or labeled product thereof according to claim 1, wherein the anion is selected from the above.

3. The zwitterionic polymer or labeled product thereof according to claim 1, wherein Z is a group selected from imidazolyl group, pyridyl group, pyridinium chloride, C1-C22 alkylpyridinium chloride, imidazolinium chloride, C1-C22 alkylimidazolinium chloride, pyridinium bromide, C1-C22 alkylpyridinium bromide, imidazolinium bromide, and C1-C22 alkylimidazolinium bromide.

4. A preservative composition for biological samples containing a zwitterionic polymer represented by the following general formula (1) or a labeled product thereof. 【Chemistry 2】 (In the formula, X 1 and X 2 These may be the same or different, and represent a carbon atom or a nitrogen atom; Y 1 and Y 2 They may be the same or different, -COO - , -SO 3 - , -OP=O(H)O - -OP=O(CH 3 ) O - and -OP = O(OR 1 ) O - The anion selected from is shown; Z represents a hydrogen atom, a C6-C10 aromatic hydrocarbon group which may be substituted with an alkyl group, a C5-C6 membered aromatic heterocyclic group which may be substituted with an alkyl group, a nitrogen-containing heterocyclic ammonium salt which may be substituted with an alkyl group, a tetraalkylammonium salt, a tetraphenylphosphonium salt, a tetraalkylphosphonium salt, a trialkylsulfonium salt, or a C1-C22 linear or branched alkyl group which may have 1-3 oxygen atoms in its molecular chain; e and f represent integers 0 or 1, respectively; l, m, and n are numbers that represent the content ratio of each repeating unit, respectively, and satisfy the conditions 0 < l ≤ 1, 0 ≤ m < 1, 0 ≤ n < 1, and l + m + n = 1; (p, q, r, s, and t each represent integers from 0 to 6.)

5. Y 1 and Y 2 However, they may be the same or different, -COO - and -SO 3 - A preservative composition for biological samples according to claim 4, wherein the anion is selected from the above.

6. The preservative composition for biological samples according to claim 4, wherein Z is a group selected from imidazolyl group, pyridyl group, pyridinium chloride, C1-C22 alkylpyridinium chloride, imidazolinium chloride, C1-C22 alkylimidazolinium chloride, pyridinium bromide, C1-C22 alkylpyridinium bromide, imidazolinium bromide, and C1-C22 alkylimidazolinium bromide.

7. Furthermore, the preservative composition for biological samples according to claim 4 contains one or more water-soluble compounds selected from electrolytes, betaine, zwitterions, alcohols, polyhydric alcohols, and sugars.

8. Furthermore, the preservative composition for biological samples according to claim 4 contains a cell-penetrating substance.

9. A preservative composition for biological samples according to any one of claims 4 to 8, which is a cryopreservative composition for biological samples, a culture composition for biological samples, a culture medium composition for biological samples, a preservation composition for biological samples, a function maintenance composition for biological samples, or a function test composition for biological samples.

10. A solvent for poorly soluble substances comprising a zwitterionic polymer or a labeled product thereof as described in any one of claims 1 to 3.

11. A method for preserving a biological sample, characterized by contacting the biological sample with a composition containing a zwitterionic polymer or a labeled product thereof as described in any one of claims 1 to 3.

12. The method for preserving a biological sample according to claim 11, wherein the composition containing the zwitterionic polymer or a labeled product thereof according to any one of claims 1 to 3 further contains one or more water-soluble compounds selected from electrolytes, betaine, zwitterions, alcohols, polyhydric alcohols, and sugars.

13. The method for preserving a biological sample according to claim 11, wherein the composition containing the zwitterionic polymer or a labeled product thereof according to any one of claims 1 to 3 further contains a cell-penetrating substance.

14. The method for preserving a biological sample according to claim 11, wherein the preservation of the biological sample is cryopreservation of the biological sample, culture of the biological sample, culture medium for the biological sample, maintenance of the function of the biological sample, or functional testing of the biological sample.

15. A method for dissolving a poorly soluble substance, characterized by dissolving the poorly soluble substance in a composition containing a zwitterionic polymer or a labeled product thereof as described in any one of claims 1 to 3.

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